The transition state for NAD+ (oxidized nicotinamide adenine dinucleotide) hydrolysis by the cholera toxin A1 polypeptide (CTA) has been characterized by multiple V/K kinetic isotope effects (KIEs) using labeled NAD+ as the substrate. CTA causes cholera by catalyzing the ADP-ribosylation of the signal-transducing G(sα) protein. In vitro, CTA catalyzes the ADP-ribosylation of several simple guanidino compounds as well as the slow hydrolysis of NAD+ (k(cat) = 8 min-1, K(m) = 14 mM) to form ADP-ribose and nicotinamide. KIEs for NAD+ hydrolysis are the following: primary 14C = 1.030 ± 0.005, primary 15N = 1.029 ± 0.004, α-secondary 3H = 1.186 ± 0.004, β-secondary 3H = 1.108 ± 0.004, γ-secondary 3H= 0.986 ± 0.003, δ-secondary 3H = 1.020 ± 0.003, and primary double = 1.052 ± 0.004. On the basis of steady-state kinetic parameters for CTA-catalyzed NAD+ hydrolysis, as well as a comparison with KIEs measured for NAD+ solvolysis, the enzymatic KIEs are near-intrinsic and describe a transition state that is relatively desolvated at the reaction center. The inability of CTA to catalyze NAD+ methanolysis is also consistent with desolvation at the reaction center. Together with the observation that CTA catalyzes ADP-ribosylation with inversion of configuration at the anomeric carbon (Oppenheimer, N. J. J. Biol. Chem. 1978, 255, 4907-4910), NAD+ hydrolysis by CTA is best described by a concerted displacement mechanism involving an enzyme-directed water nucleophile. The small, inverse solvent deuterium KIE demonstrates that a rate-limiting proton transfer does not characterize the CTA reaction coordinate. Using bond-energy bond-order vibrational analysis, the KIEs for NAD+ hydrolysis by CTA have been used to model a transition state geometry. The model is consistent with a highly dissociative, concerted mechanism, characterized by distances from the anomeric carbon to the leaving group and incoming nucleophile of approximately 2.2 and 3.3 Å, respectively. There is significant oxocarbonium ion character and hyperconjugation within the ribose ring. The γ- and δ-secondary KIEs are evidence for enzyme-substrate interactions that are remote from the reaction center and are unique to enzymatic stabilization of the transition state.
|Original language||English (US)|
|Number of pages||11|
|Journal||Journal of the American Chemical Society|
|State||Published - Jan 8 1997|
ASJC Scopus subject areas
- Colloid and Surface Chemistry